Expression of mutant and wild-type TIMP3 in primary gingival fibroblasts from Sorsby's fundus dystrophy patients

Abstract

Gingival fibroblast cell lines were derived from Sorsby's fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations. These cell lines were grown in culture to study expression of the wild-type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3) alleles from a normal diploid cell type. Firstly, patient cells were found to co-express the wild-type and mutant TIMP3 alleles, S181C TIMP3 or E139X TIMP3, at the mRNA level using restriction fragment length polymorphism (RFLP) analysis. A SpeI RFLP for E139X TIMP3 is described. Low levels of endogenous TIMP3 protein expression were elevated using the natural polysaccharide calcium pentosan polysulfate (CaPPs) in combination with the cytokine IL-1alpha. Immunoblotting detected protein expression from both wild-type and mutant alleles, S181C TIMP3 or E139X TIMP3. S181C TIMP3 from these cells was found to dimerise and retain MMP2 inhibitory activity. To facilitate studies of the E139X TIMP3 protein, the allele was expressed using HighFive insect cells. In this cell type, the E139X TIMP3 was synthesised as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in gelatin zymography. Expression of mutant E139X or S181C TIMP3 protein from a normal diploid patient-derived fibroblast cell had no effect on either MMP2 or MMP9 expression or activation whilst transcribed from their normal promoter context.

Oncostatin M in combination with tumour necrosis factor induces a chondrocyte membrane associated aggrecanase that is distinct from ADAMTS aggrecanase-1 or -2

“…42 Recombinant human TIMP-1 and TIMP-3, and native human TIMP-2 were produced inhouse. [43][44][45] Primers and probes for human glyceraldehyde-3phosphate dehydrogenase (GAPDH) and bovine ribosomal 18S were purchased from Applied Biosystems (Warrington, UK), and all other oligonucleotides were obtained from Sigma (Poole, UK). Chondroitinase ABC (catalogue No 320301) and keratanase (catalogue No 320321) were purchased from ICN Biomedicals Ltd (Thame, UK).…”

Sorsby fundus dystrophy: Insights from the past and looking to the future

“…Likewise, TIMP3 turnover is reduced in ARPE19 cells overexpressing p.(Ser179Cys), p.(Glu162*) and [(p.(Ser204Cys)] TIMP3 mutants, but remains unaffected in mouse fibroblasts expressing the p.(Ser179Cys) TIMP3 mutant. Similarly, several studies on distinct TIMP3 variants in several cell lines (BHK, COS7, ARPE‐19, patient‐derived fibroblasts) have demonstrated that mutant TIMP3 maintains its MMP2/MMP9 inhibitory function (Arris et al, ; Langton et al, ; Saihan et al, ; Yeow et al, ). In contrast, overexpression of (p.(Ser179Cys) and p.(Glu162*) TIMP3 mutants in COS7, BHK, endothelial cell, mouse fibroblast and ARPE19 cells has yielded contradictory results with respect to the retention of TIMP3’s MMP inhibitory activity (Arris et al, ; Langton et al, ; Qi et al, ; Soboleva et al, ; Yeow et al, ).…”

“…COS7, BHK, MCF‐7). Furthermore, fibroblasts obtained from human SFD patients and TIMP3 mutant mouse model have also been utilized to study the impact of TIMP3 mutation on TIMP3 expression and/or function (Arris et al, ; Soboleva, Geis, Schrewe, & Weber, ). Specific TIMP3 expression and functional attributes have been evaluated in studies comparing WT versus mutant TIMP3, including the level of TIMP3 glycosylation, secretion and aggregation/dimerization of TIMP3 in the extracellular matrix (ECM), TIMP3’s competence to bind ECM proteins like LAM and COL1 and TIMP3’s ability to inhibit specific matrix metalloproteinases or MMPs (e.g.…”

“…Interestingly, the impact of TIMP3 mutation(s) on TIMP3 expression and/or function has been cell‐type and/or mutation dependent. For instance, mutant TIMP3 [(p.(Ser204Cys)] has been shown to retain normal glycosylation pattern in SFD patient‐derived fibroblasts and BHK cells (Arris et al, ; Soboleva et al, ; Yeow et al, ). However, [(p.(Ser179Cys)] mutant TIMP3 in endothelial cells leads to increased TIMP3 glycosylation (Qi et al, ).…”

Molecular dissection of TIMP3 mutation S156C associated with Sorsby fundus dystrophy

“…Most of these introduce a potentially unpaired cysteine residue at the C-terminus of the molecule (Weber et al, 1994;Felbor et al, 1995Felbor et al, , 1996Felbor et al, , 1997Tabata et al, 1998;Langton et al, 2000;Clarke et al, 2001;Jacobson et al, 2002). Several independent studies using SFD-TIMP3 mutants overexpressed in mammalian cell systems (Langton et al, 1998(Langton et al, , 2000(Langton et al, , 2005Yeow et al, 2002) as well as endogenous TIMP3 isolated from fibroblasts of SFD patients (Weber et al, 2002;Arris et al, 2003) or mice carrying the SFDrelated S156C mutation (Weber et al, 2002;Soboleva et al, 2003) showed that mutant TIMP3 is associated to the ECM and retains its MMP-inhibitory activity. Another feature of SFD-associated TIMP3 proteins is their tendency to run at higher molecular weights when electrophoretically separated under non-reducing conditions.…”